A novel gene named NNGR (Novel Negative Growth Regulator) has been cloned. NNGR codes for a secreted protein that harbors two zinc finger-like motifs and exhibits homology to yeast killer toxins. NNGR resides at chromosome 4q21.1-22, a region frequently deleted in esophageal and a subset of colorectal cancers. NNGR expression is either lost or decreased in 5/6 colon, 1/1 gastric and 6/7 of esophageal tumors when compared with their matching normal tissues. Most importantly, NNGR expression in NNGR non-expressing colorectal cancer cells inhibits their growth by inducing apoptosis. Thus, NNGR acts as noel growth suppressor whose expression is either lost or decreased in the primary gastrointestinal (GI) cancers. The proposed studies are aimed at the molecular and functional characterization of NNGR. Specifically, fresh frozen and paraffin-embedded tissue specimens will be utilized to investigate the expression of NNGR at the mRNA and protein levels in primary colorectal, esophageal and gastric cancers and matching normal tissues. Structural alterations in NNGR will also be investigated and a correlation between the NNGR-status and clinicopathological features will be evaluated. Studies are also proposed to elucidate the molecular mechanisms of NNGR action. Towards this end, an inducible system will be used to express exogenous NNGR into cancer cells to systematically study the biological function of the NNGR protein. To investigate the structure function relationships, site-directed mutagenesis will be used to mutate important motifs and the effect of mutations on the biological and biochemical functions of NNGR will be investigated. Cre/loxP system will be used to conditionally delete NNGR in the GI tract of mice and the effects of the absence of NNGR on the GI morphogenesis and carcinogenesis will be investigated. The long-term objectives of this proposal are to elucidate (1) the role of NNGR in pathogenesis of the GI malignancies, (2) its status as a diagnostic/prognostic marker either alone or in combination with other known markers and (3) the exact molecular mechanisms of action.